Ab5622

Mice were perfused intracardially with ice-cold PBS and then 4% paraformaldehyde under deeply anesthesia. Brains were extracted, postfixed in 4% paraformaldehyde overnight at 4 °C, transferred to a 30% sucrose solution for cryoprotection, frozen, and stored at − 80 °C. We sectioned brain samples with a cryostat (Leica). The sections were then incubated with 0.2% triton-X for 15 min at room temperature in preparation for immunohistochemistry. Immunohistochemistry was performed using anti-NeuN (Chemicon, MAB377, 1:1000), anti-MAP2 (Merck, AB5622, 1:1000), anti-c-Myc 9E10 (1:1000), anti-Myc (Cell Signaling, #06-549, 1:1000), anti-FMRP (Millipore, MAB2160, 1:1000), anti-stau1 (Rockland, 600-401-EV4, 1:1000), anti-PSD 95 (NeuroMab, 75-028, 1:500), anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic System, 135 303, 1:1000), and anti-puromycin (Millipore, MABE343, 1:1000). Fluorescence intensity was determined by densitometry on sections of the frontal cortex and hippocampus (n = 3 per genotype) using ImageJ.

Immunostaining was performed using rabbit polyclonal anti‐MAP2 (1:100; #AB5622, Merck Millipore, USA) and mouse monoclonal anti‐PSD95 (1:200; #Ab2723 Abcam, UK) antibodies. Detection was performed using rabbit FITC‐conjugated (1:200; #111–095‐045, Jackson ImmunoResearch Laboratories, Inc., USA) and mouse Cy3‐conjugated (1:200; #115–165‐062, Jackson ImmunoResearch Laboratories, Inc., USA) secondary antibodies. Fluorescent images were acquired using a Nikon Eclipse TE600 microscope equipped with a Nikon Digital Camera DXM1200 ATI System (Nikon Instruments, Inc., USA). Neurite outgrowth was analyzed using the image analysis system Image‐Pro Plus as previously described.³¹ The degree of synaptic innervation was evaluated by counting the number of PSD95+ puncta on proximal dendrites and expressed as the number of PSD95+ puncta per 20 μm of length. A total of 30–40 neurons for each condition was evaluated.

Samples were immunostained based on a protocol by Taylor et al.^{26 (link)} The samples were fixed using 4% paraformaldehyde (1.00496, Sigma Aldrich) for 30 min at room temperature. The samples were then washed twice with PBS for 5 min, followed by a permeabilization step using PBS with 0.2% Triton X-100 (X100, Sigma Aldrich) for 30 min at room temperature. Non-specific binding was blocked by incubating the samples with 0.2% Triton-X and 3% BSA (A7906, Sigma Aldrich) in PBS for 2 h at 37 °C. Samples were then incubated overnight at 4 °C with a solution of PBS containing 0.2% Triton-X, 3% BSA and the primary antibodies: rat anti-MAP2 (1 : 1000, AB5622, Merck Millipore) and mouse anti-PSD-95 (1 : 1000, MA1-045, Thermo Fisher). We then rinsed the samples three times with PBS for 5 min, before incubating them with the secondary antibody solution in PBS for 1 h at room temperature. The secondary antibody solution consisted in 2 μM of Hoechst 33342, goat anti-rabbit Alexa Fluor 594 (1 : 800, A11037, Thermo Fisher) and goat anti-mouse Alex Fluor Plus 647 (1 : 800, A327728, Thermo Fisher). The samples were rinsed three times with PBS and left in PBS for imaging.

Cell cultures were fixed in 4% paraformaldehyde (100503, VWR) in PBS at room temperature (RT) for 20 min, then blocked for 1 hour in 10% normal goat serum (GS) (G9023, Sigma Aldrich) and 0.1% Triton X-100 (X100, Sigma Aldrich) in PBS. Cells were incubated with primary antibodies for 3 hours at RT. Secondary antibodies were applied for 1 hour in blocking buffer at RT. The primary antibodies used in this study were raised against rabbit ISL1 (ab109516, Abcam), rabitt-anti-Ki67 (ab15580, Abcam), rabbit-anti-GABA (A2052, Sigma Aldrich), mouse-anti-SMI32 (801701, BioLegend), rabbit-anti-MAP2 (AB5622, EMD Millipore), rabbit-anti-TUJ-1 (MRB-435P, BioLegend) Secondary antibodies were donkey and goat AlexaFluor488, 546, 555, and 650-conjugated antibodies raised against the appropriate species (used at 1:1000, Invitrogen). Where stated, the lipophilic membrane dye (FM 4-64FX, F34653, Molecular Probes) was used following the manufacturer’s instructions. Immunocytochemical images were acquired with an automated microscope (PerkinElmer Operetta), quantitative image analysis was performed using Columbus software (PerkinElmer) and Harmony High-Content Imaging software (PerkinElmer). Confocal images were acquired with a LSM 700 Inverted Confocal Microscope.

The following primary antibodies were used: anti-FLAG^®M2 (clone M2, F1804; Sigma-Aldrich Inc., St Louis, MO, USA), anti-microtubule-associated protein 2 (MAP2) (AB5622, Merck Millipore Corporation., Darmstadt, Germany), anti-postsynaptic density protein 95 (PSD95) (MAB1598, Merck Millipore), anti-SH3 and multiple ankyrin repeat domains 3 (Shank3) (sc-30193, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Synaptotagmin (ab77314, Abcam, Cambridge, UK) anti-Synaptophysin (MAB5258, Merck Millipore), anti p-Trk (sc-8058; Santa Cruz), anti-GGTase-Iβ (sc-1899, Santa Cruz), anti-TrkB (sc-12, Santa Cruz) and anti-β-actin (A5541, Sigma). Geranylgeranyl pyrophosphate (GGPP) ammonium salt, Triton™ X-100, Tween^® 20, IGEPAL^®, dimethyl sulfoxide (DMSO), filipin III, 24S-hydroxycholesterol and the chemical inhibitors GGTi-2133 and K252a were all purchased from Sigma. The phosphatase inhibitor cocktail (04906837001) and the protease inhibitor cocktails 1 and 2 (11697498001 and 11836170001, respectively) were purchased from Roche Diagnostics (GmbH, Penzberg, Germany). The fluorogenic substrate used for GGTase-I activity assay was dansyl-GCVLL (Biosynthesis, Lewisville, TX, USA). Phalloidin was obtained from Molecular Probes^®, Thermo Fisher Scientific Inc. (Waltham, MA, USA).